The invention relates to regulators of heterotrimeric G-protein mediated events and uses thereof to mediate cell signalling and membrane trafficking.
The heterotrimeric guanine nucleotide binding proteins (G proteins) are intracellular proteins best known for their role as transducers of binding by extracellular ligands to seven transmembrane receptors (7-TMRs) located on the cell surface. Individual 7-TMRs have been identified for many small neurotransmitters (e.g. adrenaline, noradrenaline, dopamine, serotonin, histamine, acetylcholine, GABA, glutamate, and adenosine), for a variety of neuropeptides and hormones (e.g. opioids, tachykinins, bradykinins, releasing hormones, vasoactive intestinal peptide, neuropeptide Y, thyrotrophic hormone, leutenizing hormone, follicle-stimulating hormone, adrenocorticotropic hormone, cholecystokinin, gastrin, glucagon, somatostatin, endothelin, vasopressin and oxytocin) as well as for chemoattractant chemokines (C5a, interleukin-8, platelet-activating factor and the N-formyl peptides) that are involved in immune function. In addition, the odorant receptors present on vertebrate olfactory cells are 7-TMRs, as are rhodopsins, the proteins that transduce visual signals.
Ligand binding to 7-TMRs produces activation of one or more heterotrimeric G-proteins. A few proteins with structures that are dissimilar to the 7-TMRs have also been shown to activate heterotrimeric G-proteins. These include the amyloid precursor protein, the terminal complement complex, the insulin-like growth factor/mannose 6-phosphate receptor and the ubiquitous brain protein GAP-43. Dysregulation of G-protein coupled pathways is associated with a wide variety of diseases, including diabetes, hyperplasia, psychiatric disorders, cardiovascular disease, and possibly Alzheimer's disease. Accordingly, the 7-TMRs are targets for a large number of therapeutic drugs: for example, the .beta.-adrenergic blockers used to treat hypertension target 7-TMRS.
Unactivated heterotrimeric G-proteins are complexes comprised of three subunits, G.alpha., G.beta. and G.gamma.. The subunits are encoded by three families of genes: in mammals there are at least 15 G.alpha., 5 G.beta. and 7 G.gamma. genes. Additional diversity is generated by alternate splicing. Where it has been studied, a similar multiplicity of G-proteins has been found in invertebrate animals. Mutations within G.alpha. subunit genes is involved in the pathophysiology of several human diseases: mutations of G.alpha. that activate Gs or Gi2 are observed in some endocrine tumors and are responsible for McCune-Albright syndrome, whereas loss-of-function mutations of G.alpha.s are found in Albright hereditary osteodystrophy.
The G.alpha. subunits have binding sites for a guanine nucleotide and intrinsic GTPase activity. This structure and associated mechanism are shared with the monomeric GTP-binding proteins of the ras superfamily. Prior to activation the complex contains bound GDP: G.alpha.GDP.beta..gamma.. Activation involves the catalyzed release of GDP followed by binding of GTP and concurrent dissociation of the complex into two signalling complexes: G.alpha.GTP and .beta..gamma.. Signalling through G.alpha.GTP, the more thoroughly characterized pathway, is terminated by GTP hydrolysis to GDP. G.alpha.GDP then reassociates with .beta..gamma. to reform the inactive, heterotrimeric complex.
The mammalian G-proteins are divided into four subtypes: Gs, Gi/Go, Gq and G12. This typing is based on the effect of activated G-proteins on enzymes that generate second messengers and on their sensitivity to cholera and pertussis toxin. These divisions also appear to be evolutionarily ancient: there are comparable subtypes in invertebrate animals. Members of two subtypes of G-proteins control the activity of adenylyl cyclases (ACs). Activated Gs proteins increase the activity of ACs whereas activated Gi proteins (but not Go) inhibit these enzymes. Gs proteins are also uniquely activated by cholera toxin. ACs are the enzymes responsible for the synthesis of cyclic adenosine monophosphate (cAMP). cAMP is a diffusible second messenger that acts through cAMP-dependent protein kinases (PKAs) to phosphorylate a large number of target proteins. Members of two subtypes, all Gi/Go proteins and the Gq proteins, increase the activity of inositol phospholipid-specific phospholipases (IP-PLCs). The activity of the subtypes are distinguishable: activation of Gi and Go are blocked by pertussis toxin whereas Gq is resistant to this compound. IP-PLCs release two diffusible second messengers, inositol triphosphate (IP.sub.3) and diacylglycerol (DAG). IP.sub.3 modulates intracellular Ca.sup.2+ concentration whereas DAG activates protein kinase Cs (PKCs) to phosphorylate many target proteins. The second messenger cascades allow signals generated by G-protein activation to have global effects on cellular physiology.
Activation of G proteins frequently modulate ion conductance through plasma membrane ion channels. Although in some cases these effects are indirect, as a result of changes in second messengers, G-proteins can also couple directly to ion channels. This phenomenon is known as membrane delimited modulation. The opening of inwardly rectifying K channels by activated Gi/Go and of N and L type Ca channels by Gi/Go and Gq are commonly observed forms of membrane delimited modulation.
Heterotrimeric G proteins appear to have other cellular roles, in addition to transducing the binding of extracellular ligands. Analysis of the intracellular localization of the various G-protein subunits combined with pharmacological studies suggest, for example, that G proteins are involved in intracellular membrane trafficking. Indeed, some workers hypothesize that G proteins evolved to control membrane trafficking and that their role in transducing extracellular signals evolved later. Studies implicate heterotrimeric G-proteins in the formation of vesicles from the trans-Golgi network, in transcytosis in polarized epithelial cells and in the control of secretion in many cells, including several model systems relevant to human disease: mast cells, chromaffin cells of the adrenal medulla and human airway epithelial cells. Nonetheless, the G-protein subunits involved in membrane trafficking and secretion have yet to be definitively established and the mechanisms by which they are activated and control membrane trafficking remains largely unknown.
Caenorhabditis elegans (reviewed in Wood, et al. (1988) The Nematode Caenorhabditis elegans. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) is a small free-living nematode which grows easily and reproduces rapidly in the laboratory. The adult C. elegans has about 1000 somatic cells (depending on the sex). The anatomy of C. elegans is relatively simple and extremely well-known, and its developmental cell lineage is highly reproducible and completely determined. There are two sexes: hermaphrodites that produce both eggs and sperm and are capable of self fertilization and males that produce sperm and can productively mate with the hermaphrodites. The self fertilizing mode of reproduction greatly facilitates the isolation and analysis of genetic mutations and C. elegans has developed into a most powerful animal model system. In addition, C. elegans has a small genome (.about.10.sup.8 base pairs) whose sequencing is more advanced than that of any other animal.
Genes that encode G-protein subunits in C. elegans were identified using probes to sequences conserved in corresponding mammalian genes. So far six G.alpha. genes have been identified including the nematode homologs of mammalian G.alpha.s, G.alpha.o and G.alpha.q/11 as well as three putative G.alpha. proteins that have not yet been assigned to a mammalian subtype class. G.alpha.o, is encoded by the gene goa-1. The G.alpha.o protein from C. elegans is 80-87% identical to homologous proteins from other species. Mutations that reduce the function of goa-1 cause behavioral defects in C. elegans including hyperactive locomotion, premature egg-laying, inhibition of pharyngeal pumping, male impotence, a reduction in serotonin-induced inhibition of defecation and reduced fertility. Mutations of goa-1 homologous to the known activating mutations of mammalian G.alpha.s and G.alpha.i2 or overexpression of wild type goa-1 caused behavioral defects which appear to be opposite to those conferred by reducing goa-1 function: sluggish locomotion, delayed egg-laying and hyperactive pharyngeal pumping.
egl-10 is a gene from C. elegans, originally identified by mutations that cause defects in egg-laying behavior (C. Trent, N. Tsung and H. R. Horvitz (1983) Genetics 104:619-647). The egg-laying defect appears to involve a pair of serotonergic motor neurons (the HSN cells) which innervate vulva muscles in C. elegans hermaphrodites (C. Desai, G. Garriga, S. L. McIntire and H. R. Horvitz (1988) Nature 336:638-646; C. Desai and H. R. Horvitz (1989) Genetics 121:703-7212).